Abstract

Recombinant neuronal nitric-oxide synthase (nNOS) expressed in baculovirus-infected Sf9 cells contains approximately 1 equiv of tightly bound tetrahydrobiopterin (BH4) per dimer and binds a second equivalent with a dissociation constant in the 10(-7)-10(-6) M range. Less is known about the pterin-binding properties of nNOS originating from expression systems such as Escherichia coli that do not produce BH4. We determined the binding properties of E. coli-expressed nNOS for BH4 and several inhibitory pterins by monitoring their effects on enzyme activity. E. coli-expressed nNOS as isolated was activated by BH4 monophasically with EC50 ≈ 2 × 10(-7) M, demonstrating a lack of tight pterin binding. However, overnight incubation with BH4 resulted in tight binding of one BH4 per dimer, yielding an enzyme that resembled Sf9-expressed nNOS. Tight pterin binding was also induced by preincubation with 4-amino-tetrahydrobiopterin, but not by 7,8-dihydrobiopterin or 4-amino-dihydrobiopterin, suggesting that tight-binding site formation requires preincubation with a fully reduced pteridine. Kinetic experiments showed that tight-binding site formation takes approximately 10 min with 1 μM BH4 (2 min with 1 μM 4-amino-BH4) at 4 °C. Anaerobic preincubation experiments demonstrated that O2 is not involved in the process. Gel electrophoretic studies suggest that tight-binding site formation is accompanied by an increase in the strength of the NOS dimer. We propose that incubation of pterin-free nNOS with BH4 creates one tight pterin-binding site per dimer, leaving the other site unaffected, in a reaction that involves redox chemistry.

Kinetics of tight binding site formation for ECo-nNOS. Shown is the effect of the time interval between BH4 and 4-amino-BH4 administration on the rate of NO formation by ECo-nNOS, as measured with the oxyHb assay. Closed circles: the enzyme was preincubated with 1 μM 4-amino-BH4 for the indicated time, after which the activity was determined in the presence of 10 μM BH4. The curve through the data points is the best fit to a single exponential with parameters: kobs = 1.6 ± 0.3 min–1, Act0 = 151 ± 4 nmol·mg–1·min–1, Act∞ = 87 ± 2 nmol·mg–1·min–1. Open circles: the enzyme was preincubated with 1 μM BH4 for the indicated time, after which the activity was determined in the presence of 10 μM 4-amino-BH4. The curve through the data points is the best fit to a single exponential with parameters: kobs = 0.23 ± 0.10 min–1, Act0 = 16 ± 4 nmol·mg–1·min–1, Act∞ = 79 ± 11 nmol·mg–1·min–1. In both cases data points are shown ± SEM (n = 3). Preincubation conditions: 2 μg/mL (∼11 nM) ECo-nNOS, 0.2 mM Arg, 0.2 mM NADPH, 5 μM FAD, 5 μM FMN, 0.5 mM CaCl2, 5 μM oxyHb, 1000 U/mL SOD, 50 mU/mL CAT, 0.2 mM CHAPS, 0.1 mM EDTA, 50 mM TEA (pH 7.4), and 1 μM BH4 or 4-amino-BH4 at 4 °C. The assay conditions were the same except for the temperature (37 °C), the presence of 10 μM 4-amino-BH4 or BH4 (in the case of preincubation with BH4 and 4-amino-BH4, respectively), and the presence of 10 μg/mL CaM, which was added to start the reaction. In both cases, data points are shown ± SEM (n = 3).

Effect of pteridine preincubation on ECo-nNOS dimer stability. ECo-nNOS was preincubated with or without pteridines (BH4, BH2, 4-amino-BH4, or 4-amino-BH2) and Arg, followed by LT-PAGE. Shown is the relative content of SDS-resistant dimer, estimated from densitometric analysis. Preincubation conditions: 20 μg of ECo-nNOS in 0.1 mL of TEA (50 mM, pH 7.4) and 0.2 mM pteridines and 0.1 mM Arg as indicated, for 5 min at 37 °C. See experimental procedures for further details. 4AH4: 4-amino-BH4; 4AH2: 4-amino-BH2.

Dimeric nNOS contains two identical sites that bind BH4 with relatively low affinity, manifested by a EC50 of ∼0.2 μM in the activity assays. Binding of the first equivalent of BH4 causes a redox reaction that results in very tight binding of the pterin. The reaction may involve a unique site on the enzyme (the green dot in the scheme) that is not too far away from both binding sites, perhaps the Zn-binding site at the dimer interface. Upon tight binding of 1 equiv of BH4, this site is no longer able to assist in tight binding of the second equivalent of BH4 (illustrated in the scheme by the change in color from green to red). Consequently, only 1 equiv of BH4 is tightly bound, with the other site still exhibiting EC50 ≈ 0.2 μM. The radioligand binding studies suggest that BH4 binding is fully reversible, indicated by the short backward arrow in the middle of the scheme. However, it is presently unclear if the phenomenon of tight binding is covered by the radioligand binding studies under the applied conditions. It is also unclear if dissociation of tightly bound BH4, if it occurs, is accompanied by irreversible inactivation and/or changes in the quaternary structure of the enzyme.